Abstract
Purpose :
Mitochondrially Derived Peptides (MDPs) are short peptides encoded off the short open reading frames (sORF) of human mitochondrial DNA. Humanin (HN), a 24 amino acid MDP has both in vitro and in vivo benefits improving cell survival. Humanin-G (HNG) has a serine-to-glycine substitution at position 14, making it a 1000-fold more potent variant. We hypothesized that subconjunctival injection of HNG loaded microspheres (HNG-MS) would improve retinal function and alter gene expression in both RPE and retina of treated RCS rats.
Methods :
21 day old RCS rats received subconjunctival injections in both eyes of (1) 286μg Empty-MS in 100μL Buffered Saline Solution (BSS) (N=6); (2) 286μg HNG-MS in 100μL BSS (N=5); or (3) received no injection (N=6). Functional Analyses. Scotopic & Photopic Threshold Response Electroretinography (ERGs) was performed at 14 and 27 Days After Starting Treatment (DAST). Molecular Analyses. Animals were sacrificed after 4 weeks. One eye was used for immunohistochemistry (IHC), and the other for eye for RNA extraction and gene expression analysis (qRT-PCR). Statistical analysis was performed using Graphpad Prism, using Mann-Whitney U. The significance set at p<0.05.
Results :
Subconjunctival injections of HNG-MS significantly improved the ERG Scotopic A-wave (166% increase, P=0.0057) compared to the Empty-MS treated RCS rats. The Scotopic B-wave trended towards improvement but did not reach significance (59% increase, P=0.0668). (HNG-MS, n=4; Empty-MS, n=6; NT (n=2). (Fig.1) At 30 DAST with HNG-MS, in neuroretinal cells there was decreased expression of inflammation genes (TNFa 0.53-fold, P=0.032; IL1B, 0.47-fold, P=0.009; CCL2, 0.64-fold, P=0.016) (Fig. 2); In RPE cells, there was decreased expression of apoptosis genes (DDIT3, 0.78-fold, P=0.03; CASP3, 0.7-fold P=0.016; CASP7, 0.77-fold, P=0.029; CASP9, 0.77-fold P=0.016) (Fig. 3), along with antioxidant (SOD2, 0.64-fold P=0.016) and inflammatory genes (TNFa, 0.11-fold, P=0.029; IL1B, 0.25-fold, P=0.004) (Fig. 4). Significance of gene expression values for HNG-MS compared with Empty-MS.
Conclusions :
The subconjunctival injection of HNG-MS provides significant improvement in retinal function and beneficial impact on gene expression in RPE and neuroretina of RCS rats. This supports our hypothesis that HNG loaded microspheres can serve as an effective delivery method for HNG in models of retinal degeneration.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.